Development and validation of a nonisotopic immunoassay for the detection of LSD in human urine.

A microplate enzyme immunoassay (EIA) for the detection of lysergic acid diethylamide (LSD) in human urine was developed. The assay kit is designed around an LSD derivative coated on the wall of microplate wells with preservatives and stabilizers. Sample and rabbit anti-LSD are added to the microplate well. The immobilized LSD and LSD present in specimens compete for the opportunity to bind to the anti-LSD antibodies. An anti-rabbit antibody labeled with horseradish peroxidase is used to provide the assay signal, which is inversely proportional to the concentration of LSD in the sample. The assay requires a 25-microL urine sample and three consecutive incubation periods of 60, 30, and 30 min at room temperature. The assay was tested with a variety of drugs, including ergot alkaloids spiked into drug-free urine at up to 100,000 ng/mL without cross-reaction. Nor-LSD was shown to cross-react between 16% and 28%, depending on its concentration. Of the other compounds tested, only ergonovine demonstrated slight cross-reactivity at approximately 0.0008%. The assay is designed to be used with a qualitative cutoff of 0.5 ng/mL. Precision testing at 0.5 ng/mL gave a coefficient of variation (CV) of 6% based on 20 replicates. The CV at 0.375 ng/mL (cutoff, -25%) was 5.2% and at 0.625 ng/mL was 6.6%. Precision at other concentrations within the range of the calibration curve gave similar results both intra- and interassay. Clinical performance of the assay was compared with that of a commercial radioimmunoassay (RIA). Comparable performance was observed with both methods, each screening a total of 458 samples as negative and 17 samples as positive relative to a 0.5 ng/mL cutoff. The EIA found an additional three positive samples that were negative by RIA. The EIA is suitable for the screening of urine samples for the presence of LSD. Preliminary indications are that the assay is also suitable for use with whole blood specimens. The assay can be performed manually or be fully automated and without the need for radioactivity; it can be used in any laboratory.

Morphine and morphine-6-glucuronide plasma concentrations and effect in cancer pain.

The relationships between plasma morphine and metabolite (M3G and M6G) concentrations and analgesic efficacy were investigated in an open study of 39 cancer pain patients receiving chronic oral morphine therapy with either morphine sulfate solution or controlled-release morphine tablets. There were no differences in morphine, metabolite kinetics, or analgesic efficacy between equivalent doses of conventional or controlled-release formulations. The increase in morphine plasma concentration after a dose (1 hr for normal release, 2 hr for controlled release) was correlated significantly with the dose of morphine (r = 0.914, P < 0.001). There was a significant reduction in pain intensity (P < 0.05) and increase in pain relief (P < 0.001) after the dose of morphine administration, when compared with the predose score. One-half of the patients had mild and tolerable adverse effects. Patients were classified by mean pain relief between doses as having optimal, moderate, or poor pain control. No simple relationship was found between morphine plasma concentration and pain control. Morphine plus M6G concentrations in the "optimal control" group (751.2 +/- 194 nmol/L), however, were more than twice those found in the "moderate control" group (276.9 +/- 41.9 nmol/L) (P < 0.05), and no patient in the moderate control group had a morphine plus M6G concentration greater than 405 nmol/L. These results support the importance of M6G in morphine analgesia. For these hospitalized patients, there appeared to be a therapeutic range of morphine plus M6G plasma concentrations for optimal pain control with a lower limit of 400 nmol/L predose.

Epithelial mucin core antigen (EMCA) in assessing therapeutic response in advanced breast cancer--a comparison with CA15.3.

We report a comparative study of CA 15.3 and EMCA (epithelial mucin core antigen) in 77 consecutive women with newly diagnosed UICC assessable metastatic breast cancer, 59 patients received hormones and 18 chemotherapy. Assessments of response were made prior to commencing therapy and repeated 2 monthly. Sites of metastatic disease included bone (34), pulmonary (8), bone and pulmonary (14) and visceral (21). Using a cut-off of 33 U ml-1 changes in EMCA at 2, 4 and 6 months showed a highly significant correlation (P < 0.001) with UICC assessed response at 6 months; selectivity 70%, sensitivity 80%, specificity 91%, positive predictive value 84%; negative predictive value 89% at 2 months. Corresponding values for CA 15.3: selectivity 89%, sensitivity 85%, specificity 91%, PPV 92% and NPV 91%. Four of eight patients unassessable by CA 15.3 were assessable by EMCA; four patients expressed neither marker. EMCA appears to reflect tumour bulk and may be useful in monitoring therapy in patients with advanced breast cancer. With an easier and more robust assay format than CA 15.3, EMCA is potentially a more useful marker.

The mucin antigens: what are we measuring?

Serum of breast cancer patients contains high molecular weight, mucin-like glycoproteins which are held to be differentiation markers for certain types of normal epithelia, in particular mammary epithelium. These components have primarily been identified using monoclonal antibodies raised against human milk fat globule membranes, tumour extracts or purified mucins. Even so, many of the antibodies produced react with a discrete region of the mucin protein core involving the hydrophilic turn domain APDTRPAP. The present investigation using the anti-urinary mucin antibody, C595, illustrates both the clinical potential of the mucin antigens in breast cancer studies as well as the exquisite specificity of immune recognition of a complex polymorphic glycoprotein at the level of the individual amino acids.

Oral morphine in cancer pain: influences on morphine and metabolite concentration.

One hundred fifty-one patients with chronic cancer pain were studied during chronic treatment with oral morphine. Plasma concentrations of morphine and metabolites (M3G and M6G) were measured. The ratio of plasma morphine to metabolites was not affected by dose. Generalized linear interactive modeling analysis using morphine dose, age, sex, renal and hepatic dysfunction, and concomitant medication as explanatory variables accounted for 70% of the variance in plasma concentrations of morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). Increasing morphine dose was a significant factor for increased plasma concentrations of morphine, M3G, and M6G. Other significant factors were: age greater than 70 years (increased M3G and M6G plasma concentrations), plasma creatinine greater than 150 mumol/L (increased M3G and M6G plasma concentrations), male sex (decreased morphine and M6G plasma concentrations), raised creatinine plus coadministration of tricyclic antidepressants (increased M3G plasma concentrations), ranitidine (increased morphine plasma concentrations), and raised creatinine plus coadministration of ranitidine (increased M6G plasma concentrations).

Buprenorphine disposition in patients with renal impairment: single and continuous dosing, with special reference to metabolites.

The disposition of buprenorphine has been studied in two patient groups to assess the influence of impaired renal function on the metabolism of buprenorphine and two of its metabolites, buprenorphine-3-glucuronide (B3G) and norbuprenorphine (NorB). A single i.v. dose of 0.3 mg was given to 15 patients (nine with dialysis-dependent renal failure) undergoing lower abdominal or peripheral body surface surgery. Blood was sampled up to 24 h. Concentrations of buprenorphine, B3G and NorB were assayed by a differential radioimmunoassay technique. There were no differences in buprenorphine kinetics between anaesthetized healthy patients and those with renal impairment: mean elimination half-lives 398 and 239 min; clearance 651 and 988 ml min-1; apparent volume of distribution at steady state 313 and 201 litre, respectively. Both metabolites were undetectable following the single i.v. dose. In a second group of 20 patients (eight with renal impairment), buprenorphine was administered by continuous infusion for provision of analgesia and control of ventilation in the ITU (median infusion rate 161 micrograms h-1 (range 36-230 micrograms h-1) for a median duration of 30 h (2-565 h). Buprenorphine clearance in patients with normal and impaired renal function was similar (934 and 1102 ml min-1, respectively), as were dose-corrected plasma concentrations of buprenorphine. In patients with renal failure, plasma concentrations of NorB were increased by a median of four times, and B3G concentrations by a median of 15 times.

Radioimmunoassay of buprenorphine in urine: studies in patients and in a drug clinic.

A radioimmunoassay kit (DPC buprenorphine double antibody) was evaluated with clinical samples and samples from a drug clinic. Urine samples were collected over a 2-day period from 5 hospital in-patients receiving sublingual buprenorphine, 400 to 2000 micrograms/day, for the relief of chronic pain. Samples were measured before and after enzymatic hydrolysis. Urine buprenorphine concentrations were measurable at all doses studied (minimum value 5.6 ng/mL) and were greater with larger doses. The increase in concentration after hydrolysis averaged 49% and was similar for all doses studied. The authors conclude that the method has extensive cross-reactivity with glucuronides of buprenorphine and its metabolites and that samples may be analyzed without prior hydrolysis. The prevalence of buprenorphine use in 97 patients attending a drug clinic was also studied. Sixty (62%) had measurable urinary buprenorphine concentrations of 1 ng/mL or more by direct assay. The buprenorphine users were significantly younger and reported significantly greater use of opiates than nonusers.

Studies on morphine disposition: influence of general anaesthesia on plasma concentrations of morphine and its metabolites.

The kinetics of morphine were studied during balanced anaesthesia in 10 patients undergoing lower abdominal or body surface surgery, and compared with those obtained in nine awake patients receiving morphine i.v. for the relief of chronic non-cancer pain. All patients received morphine sulfphate pentahydrate 10 mg i.v. over 30 s. Venous blood samples were collected for up to 180 min, and plasma concentrations of morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) assayed by a differential radioimmunoassay technique. There were no differences between groups with respect to the elimination half-life (awake group: 207 min; anaesthetized group: 153 min), volume of distribution at steady state (awake: 147 litre; anaesthetized: 128 litre), or clearance (awake: 587 ml min-1; anaesthetized: 766 ml min-1). Peak concentrations of M3G were similar in the two groups, but the peak concentration of M6G was greater in the anaesthetized patients. The AUC for M3G and M6G (0-180 min) also were greater in the anaesthetized patients, presumably as a result of decreases in renal blood flow and glomerular filtration rate during halothane anaesthesia.

Studies on morphine disposition: influence of renal failure on the kinetics of morphine and its metabolites.

The influence of renal failure on the disposition of morphine and its metabolites was studied in nine patients with end-stage renal failure undergoing transplantation, and compared with five healthy anaesthetized patients. All patients received morphine sulphate pentahydrate 10 mg i.v. over 30 s, as part of a balanced anaesthetic technique. Venous blood samples were collected for up to 24 h, and plasma concentrations of morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) assayed by a differential radioimmunoassay method. There were no differences between the two groups for morphine elimination half-life (renal failure: 290 min; anaesthetized controls: 286 min), or clearance (renal failure: 533 ml min-1; controls 741 ml min-1). However, the volume of distribution at steady state was greater in the control group (241 litre v. 141 litre; P = 0.002). The peak concentrations of M3G and M6G were greater in the renal transplant patients (P = 0.001 and P = 0.01, respectively), as were the AUC (0-24 h) (P = 0.002 and P = 0.002). M6G has been shown to possess analgesic properties in both man and experimental animals, and therefore the increased AUC for M6G may contribute to the prolonged effect seen with morphine when given to patients with impaired renal function.

Studies on morphine disposition: plasma concentrations of morphine and its metabolites in anesthetized middle-aged and elderly surgical patients.

The effects of aging on the disposition of morphine and its metabolites have been investigated in 10 middle-aged patients (36 to 55 years of age) undergoing lower abdominal or body surface surgery, and compared with 10 elderly patients (65 to 83 years of age) undergoing similar surgery. All patients received 10 mg morphine sulphate pentahydrate IV over 30 seconds as part of a balanced anesthetic technique. Peripheral venous blood samples were collected to 180 min, and plasma concentrations of morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) were assayed by differential radioimmunoassay. There were no differences between the two groups for morphine elimination half-life (middle-aged patients, 129 min; elderly patients, 162 min), mean residence time (154 and 207 min), and apparent volume of distribution at steady state (116 and 107 l). However, clearance was significantly greater in the younger patients (853 vs. 559 ml/min; p less than 0.02). The area under the curve (AUC0-180) for M3G and M6G were similar in the two patient groups, as were the peak metabolite concentrations and times to peak concentrations. M6G has been shown in both animals and humans to exert analgesic properties. Despite the reduced clearance of the parent drug, there was an unaltered AUC for M6G, presumed due to the greater decrease in glomerular filtration rate seen during anesthesia in the elderly patient. This phenomenon may result in enhanced analgesic efficacy from a given dose of morphine in the elderly patient.

Morphine and ibuprofen compared using the cold pressor test.

The analgesic efficacy of single doses of oral morphine sulphate solution (10 mg) and ibuprofen 600 mg was compared in 12 volunteers using a double-blind, double-dummy, placebo-controlled design on the cold pressor experimental pain model. Measurement of pain intensity was made before medication and then at 30, 60, 90, 120 and 180 min; blood samples were taken at these times for measurement of morphine and glucuronide metabolites by radioimmunoassay. Sessions were at least 5 days apart. Correlations were sought between analgesic effect and plasma concentrations of either morphine or morphine-6-glucuronide. Morphine produced significant reduction in both peak pain intensity and area under the pain intensity curve compared with placebo; the threshold time was significantly increased by morphine compared with placebo. Ibuprofen was statistically indistinguishable from placebo on all three measures of analgesia. Analgesic effect and plasma concentrations of morphine showed significant correlation (P = 0.053). The study confirmed reports of the opiate sensitivity of the cold pressor model, and the apparent insensitivity of the model to non-steroidal anti-inflammatory drugs.

Comparison of whole blood and plasma morphine.

Thirty paired samples of plasma and whole hemolyzed blood were obtained from patients undergoing surgery. Morphine concentrations were measured with the DPC serum morphine kit without prior treatment. In a regression analysis, the equation to the regression line was blood = 1.02 plasma + 1.0 ng/mL, and the correlation coefficient was 0.994. Radioimmunoassay screening of postmortem blood samples can be performed without prior sample treatment.

Analysis of morphine and its major metabolites by differential radioimmunoassay.

The analysis of morphine, morphine-3-glucuronide (M-3-G) morphine-6-glucuronide (M-6-G) by differential radioimmunoassay using iodinated label and three different antisera is described. These methods were used to measure concentrations of morphine and its conjugated metabolites in human plasma, over a 3-h period, following a single 10 mg intravenous dose. In 13 patients peak concentrations of M-3-G (739 nmol/L +/- 73.7 SEM) were approximately 10 times greater than those of M-6-G (71.3 nmol/L +/- 8.6 SEM). Times to reach these peaks were similar for both metabolites. Decay of morphine from plasma followed a biexponential pattern with a mean terminal half-life of 59.3 min (+/- 8.1 SEM, n = 11). Accurate determination of the half-lives of the glucuronides was not possible due to the short sampling period, but M-6-G seemed to have a similar half-life to morphine, while M-3-G was eliminated more slowly.

Epinephrine reduces systemic absorption of extradural diacetylmorphine.

The effect of epinephrine on the vascular absorption of morphine from the extradural space is uncertain; this study examined the effect of epinephrine on the related but more lipophilic opiate diacetylmorphine (diamorphine, heroin) because any effects of vasoconstriction on diacetylmorphine absorption should be maximally apparent. With this experiment, we hoped to resolve whether epinephrine does or does not alter vascular absorption of extradurally injected opiates. Thirty patients undergoing lumbar laminectomy were given either extradural diacetylmorphine, 5 mg, extradural diacetylmorphine, 5 mg with 1:200,000 epinephrine, or 1:200,000 epinephrine followed 5 min later by 5 mg extradural diacetylmorphine. Plasma morphine concentrations were measured by radioimmunoassay because of the rapid conversion of diacetylmorphine to morphine in plasma; repeated blood samples were obtained the first 30 min after injection into the epidural space. Significantly lower plasma morphine levels occurred between 3 and 20 min when epinephrine was added to diacetylmorphine. Peak plasma morphine levels (mean +/- SEM) were 179 +/- 37 nmol/L with diacetylmorphine alone, 87 +/- 16 nmol/L with diacetylmorphine and epinephrine given together and 44 +/- 11 nmol/L with epinephrine pretreatment, all significantly different from one another. The mean peak plasma morphine concentration was 8.7 +/- 1.1 min for diacetylmorphine alone, but addition of epinephrine (together or sequentially) meant that 15 of 20 patients had no peak level before 120 min. Epinephrine reduced absorption of diacetylmorphine from the extradural site by at least 55% over the first 30 min. The incidence of patients with more than 9 hr analgesic duration was significantly (P = 0.033) greater in patients who had diacetylmorphine and epinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrathecal morphine and heroin in humans: six-hour drug levels in spinal fluid and plasma.

Lumbar spinal fluid and plasma concentrations of morphine were measured by radioimmunoassay after intrathecal administration of 1 mg of morphine (n = 13) or heroin (n = 10). Plasma levels of morphine were measured regardless of 'whether heroin or morphine was injected intrathecally, because of the rapid biotransformation of heroin to morphine in plasma. Significant drug concentrations appeared in plasma after intrathecal heroin (peak concentration 47.8 +/- 9.0 nmol/L, time to peak concentration 10 +/- 2.4 min); after intrathecal morphine plasma drug concentrations were significantly lower (8.1 +/- 1.0 nmol/L; P less than 0.002) and significantly later (216 +/- 39 min; P less than 0.002). Elimination half-life of heroin from spinal fluid (43 +/- 5 min) was significantly shorter than for morphine (73 +/- 5 min; P less than 0.02).

Radioimmunoassay of buprenorphine with iodine label: analysis of buprenorphine and metabolites in human plasma.

Quantitative analysis of potent opiate drugs in plasma by radioimmunoassay is potentially inaccurate because of the occurrence of cross-reacting metabolites. This paper describes the chemical synthesis of buprenorphine-3-O-glucuronide, a metabolite of buprenorphine, and an extraction procedure coupled with radioimmunoassay which allows the sensitive and specific measurement of buprenorphine using an iodinated buprenorphine derivative. The measurement of extracted and unextracted samples using two different antisera allowed investigation of the metabolism of buprenorphine. In four patients who had taken sublingual buprenorphine for at least one month, N-dealkyl buprenorphine was present in similar concentrations to those of buprenorphine, while buprenorphine-3-O-glucuronide was present in two to three times those concentrations.

Dural permeability to narcotics: in vitro determination and application to extradural administration.

The permeability of cranial and lumbar dura to various substances including a number of narcotic analgesics was measured in vitro. Preliminary data On human postmortem material is reported. Permeability had a linear relation to the inverse of the square root of molecular weight. This is the expected relationship for a diffusion process dependent upon molecular weight. The differential mass selectivity coefficients for lumbar and cranial dura were calculated; they were similar at 0.8 and 0.9. This was greater than for diffusion in simple liquids, but much less than that for biological lipid membranes. This suggests that the low rates of diffusion are a property of the thickness of the dura rather than any inherent impermeability. A simple model for the dural transfer of drugs is described, and applied to narcotics. Its purposes were to suggest: the factors involved in the dural transfer of drugs; the physicochemical properties of drugs relevant to their dural transfer; worthwhile measurements in future studies. The model indicates that drug molecular weight and rate of absorption are important determinants of the efficiency of dural transfer. Low molecular weight and slow absorption produce high dural transfers. When applied to narcotics, these factors could produce a difference of up to an order of magnitude in the amount transferred directly across the dura.

Infrared spectroscopy at 20,000 scans per second.

Improvements in a rapid scan ir spectrometer used for flash photolysis-kinetic spectroscopy are described. The scanning mechanism now regularly operates at a repetition rate of 20,000 scans/sec; the photolysis flash has a half-time of 7.5 microsec, with energy up to 2940 J, and at 1500 J it yields 5 x 10(18) quanta/flash; the spectroscopic light source operates with a brightness temperature of 2400 K. The relationship between spectral resolution and scanning speed is quantitatively discussed.